Long-range Atoh1 enhancers maintain competency for hair cell regeneration in the inner ear

During tissue regeneration, lineage-related cells can switch their fate to replace missing cells. This cell plasticity is particularly prominent in more regenerative vertebrates such as zebrafish, yet the molecular basis by which cells transdifferentiate into another cell type upon injury remains unclear. Here we investigate the epigenetic basis of regenerative transdifferentiation in the inner ear, where supporting cells (SCs) generate mechanosensory hair cells (HCs) upon damage. By comparing the chromatin landscapes in regenerative zebrafish and green anole lizards versus non-regenerative mice, we identified a class of enhancers that function in progenitors to generate HCs and then are selectively maintained in SCs of regenerative vertebrates to regenerate HCs. In particular, we uncovered a syntenic class of long-range enhancers for Atoh1, a master transcription factor for HC differentiation. In the absence of injury, these enhancers maintain accessibility in SCs through adulthood but are prevented from driving zebrafish atoh1a expression through Notch repression. Deletion of these enhancers not only impaired atoh1a expression and HC formation during development but also blocked the ability of SCs to transdifferentiate into HCs during regeneration. Moreover, defects were specific to the inner ear versus the lateral line, revealing distinct mechanisms of regeneration in these mechanosensory organs. These findings reveal a class of regenerative enhancer that maintains competency of inner ear SCs to upregulate atoh1a and transdifferentiate into HCs upon damage. We propose that the continued accessibility of developmental enhancers for one cell fate in lineage-related cells may be a common theme underlying adult cell plasticity in regenerative vertebrates. Overall design: Inner ears were dissected from the Sox10Cre;UbiSwitch zebrafish at 14dpf (n=40) and 12mpf (n=6) zebrafish. Converted cells were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of mCherry signal and analyzed using 10x genomics based snATACseq or multimome ATAC+Gene Expression kits. scRNA/snRNA associated with these two timepoints were previous deposted at GSE211728. Reads were aligned to danRer11 using the Cellranger pipeline. Inner ear saccules were dissected from adult green anole lizards heads (n=7, heads were 2.5-3cm) and FACS sorted for live cells by DAPI exlucsion. 10x Genomics multiome ATAC+gene expression library was constructed and reads were aligned to AnoCar2.0v2 using the cellranger pipline

在组织再生过程中，谱系相关细胞可转变细胞命运以替代缺失的细胞。这种细胞可塑性在斑马鱼等再生能力较强的脊椎动物中尤为显著，但细胞在损伤时转分化为其他细胞类型的分子基础仍不明晰。本研究探究了内耳再生性转分化的表观遗传机制：当内耳受到损伤时，支持细胞（supporting cells, SCs）可生成机械感觉毛细胞（mechanosensory hair cells, HCs）。我们通过对比具有再生能力的斑马鱼、安乐蜥与无再生能力的小鼠的染色质开放景观，鉴定出一类增强子：这类增强子在祖细胞中发挥功能以生成毛细胞，随后在再生脊椎动物的支持细胞中被选择性维持，以介导毛细胞的再生。尤为关键的是，我们发现了一类针对Atoh1的同线性长距离增强子——Atoh1是调控毛细胞分化的核心转录因子。在未受损伤的状态下，这类增强子在成年支持细胞中始终保持染色质开放状态，但通过Notch通路的抑制作用，阻止斑马鱼atoh1a基因的表达。敲除这类增强子不仅会损害发育过程中atoh1a的表达与毛细胞的形成，还会阻断再生过程中支持细胞向毛细胞转分化的能力。此外，该缺陷仅出现于内耳，而侧线系统不受影响，这揭示了这两类机械感觉器官截然不同的再生机制。本研究的发现揭示了一类再生性增强子：这类增强子可维持内耳支持细胞的潜能，使其在损伤时能够上调atoh1a基因表达并转分化为毛细胞。我们提出，在谱系相关细胞中，针对某一细胞命运的发育增强子持续保持开放状态，可能是再生脊椎动物成体细胞可塑性的共同核心机制。实验整体设计：于14天胚胎期（14dpf，n=40）和12月龄（12mpf，n=6）的Sox10Cre;UbiSwitch斑马鱼体内解剖内耳，根据mCherry信号的有无，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离已转化的细胞，并采用基于10x Genomics的单细胞核转座酶可及性测序（single-nucleus assay for transposase-accessible chromatin sequencing, snATAC-seq）或多组学ATAC+基因表达试剂盒进行分析。与这两个时间点相关的单细胞RNA测序/单细胞核RNA测序数据已预先提交至GSE211728。测序reads通过Cellranger流程比对至danRer11参考基因组。于成年安乐蜥头部（n=7，头部长度2.5-3cm）中解剖内耳球囊，通过DAPI排除法进行荧光激活细胞分选以分离活细胞。构建10x Genomics多组学ATAC+基因表达文库，测序reads通过Cellranger流程比对至AnoCar2.0v2参考基因组。